The long term objective of this research program is to understand the mechanism of gene activation in terms of chromatin structure. The specific aims for the coming year are to a) expand the available information on chromosomal proteins preferentially associated with active loci in a developmental time frame, using immunofluorescence staining techniques on polytene chromosomes; b) develop further an in vitro transcription system using Drosophila nuclei; and c) expand out information on chromatin structure and the structural alterations occurring on gene activation at specific loci using nuclease digestion techniques. Recent work on the Drosophila heat shock loci has demonstrated that specific higher order "domains" of chromatin can be revealed by limited DNase I digestion. Initial cleavage of chromatin by both DNase I and micrococcal nuclease shows some specificity in terms of a given locus. Nucleosomal organization and perhaps higher order structure are disrupted on gene activation, as shown by smearing in the pattern of fragments generated by nuclease digestion of the active loci. It is proposed to determine the generality of these observations by examining other loci. The DNase I cleavage sites and regions of structural perturbation in relation to the transcription site will be mapped, probably for 63BC, a heat shock locus.